Mass spectrometry has been widely used for the identification and quantitative analysis of proteins and peptides. For example, peptides produced from the enzymatic digestion of proteins were ionized using Matrix-Assisted Laser Desorption/Ionization (MALDI) or Electrospray Ionization (ESI) and then subjected to mass analysis to accurately measure the mass thereof and be compared with the peptide information provided by the genetic sequences to reveal the identity of proteins. More specifically, some peptide ions were selected and decomposed by collision in the mass spectrometer to obtain the sequences of peptides from fragment ions, thereby revealing the identity of proteins.
For the quantitative analysis of proteins and peptides, the method of labeling proteins and peptides to be analyzed with chemical labels comprising stable isotopes has been widely used to analyze the mass. When the same kinds of chemical labels with different isotopes were attached to the same kinds of many protein and peptide samples for quantitative comparison and subjected to mass analysis, the masses of each of the samples are different on the mass spectrum or tandem mass spectrum due to the difference in the masses of isotopes so that they cannot be analyzed simultaneously.
To simultaneously conduct the identification and quantitative analysis of proteins and peptides, chemical labeling with isobars has been employed. In US Patent Publication No. US 2005/0148087 and International Patent Publication No. WO 2005/068446 and the like, isobaric chemical labels have been disclosed designed to display quantitation signals on tandem mass spectrum through attaching to peptides and collision and decomposition. However, because the labeling agents presented in said references use isotopes such as carbon-13, nitrogen-15, or oxygen-18, etc., there are problems that the synthesis of various isobars is limited and the price is too high. Further, the quantitative analysis using isobaric labeling agents disclosed in the prior art cannot be employed in quadrupole ion trap mass spectrometer such as Paul trap, linear ion trap, etc. Thus, there is a need to provide a new isobaric labeling agent that can simultaneously identify the peptide sequence and the protein quantity using various hydrogen isotopes which can substitute for hydrogen and have a relatively low price.
Meanwhile, quadrupole ion trap mass spectrometers have been widely used to reveal the identity of proteins and peptides since they are cheaper, easy to maintain and manage, and convenient to use as compared with other mass spectrometers. They also have an ability to conduct tandem mass spectrometry several times by collecting ions in the gas phase. For this reason, quadrupole ion trap mass spectrometers have been most widely provided and managed in the field of proteome study. As a general summary, tandem mass spectrometry using such quadrupole ion traps generally consists of a technology called Resonant Excitation Collision-Induced Dissociation (RE-CID). In such a case, when the mass-to-charge ratio of fragment ions is less than about ⅓rd of the mass-to-charge ratio of parent ions, ions are not stably collected in the ion trap and thus cannot be detected. It is referred to as the ‘low-mass cutoff’ effect. The isobaric labeling agents employed in the prior technology use small fragment ions having the mass of about 100-200 Da as a quantitation signal and thus have the problem that they are not detected as a quantitation signal ion in quadrupole ion trap mass spectrometers due to the low-mass cutoff effect. Furthermore, accurate quantitative analysis may not be possible, since in the mass region of 100-200 Da there is a very great possibility that the internal fragment ions having small mass, which can be derived from the analyte proteins or peptides, interfere with the quantitation signal ions.
Thus, the materials disclosed in the above-mentioned patents can be used only for analysis of fragment ions having small mass and therefore have a critical limitation in that they cannot be used in quadrupole ion trap mass spectrometers. For this reason, the invention relating to the isobaric labels that can be used in most widely distributed quadrupole ion trap mass spectrometers without any limitation and the analytical methods using the same is needed. In addition, in order to overcome said limit the quantitation signal needs to be displayed as the fragment ions having a sufficiently high mass. Since the fragment ions having high mass have a very low possibility that they are disturbed by noise signal, as compared to the fragment ions having low mass, and are not restricted by the low-mass cutoff, which can be caused in quadrupole ion trap mass spectrometers, they have high applicable value.
Meanwhile, in Korean Patent Application No. 2008-0070272 the present inventors have disclosed the new isobaric labeling agents named Mass-balanced isotope tag (MBIT) that uses only hydrogen isotope, can control the mass of quantitation signal, and has a dipeptide structure. In addition, Korean Published Patent No. 2010-0009466, Korean Patent Publication No. 2010-0009479, and International Patent Publication No. WO 10/008,159 have proposed a mass-variable labeling agent and a set of mass-variable labeling agents, wherein the mass controlling group is modified to diversify the properties and quantitation signal mass of the isobaric labeling agents, and further provided the multiplexed quantitative analytical method using the same, that is, the multi 2-plex quantitative method, which is a simultaneous, multiplexed quantitative analytical method for three or more samples using two or more kinds of the labeling agents. Although by utilizing said method the isobaric labeling agents can be easily and inexpensively synthesized, and further, multiplexed samples can also be quantified, when the multiplexed quantification is accomplished according to the multi 2-plex quantitative method, the drawbacks are that too much of the standard samples are consumed and the total quantity of the samples to be analyzed at a time is also increased.
Thus, the present inventors have researched a labeling agent that utilizes hydrogen isotope and, at the same time, can quantify multiplexed samples at the same time as well as decreasing the cost for synthesis of the labeling agent. As a result, we have identified that such purposes can be achieved through anew chemical structure, thereby completing the present invention. In addition, even in cases where the multiplexed samples are analyzed, we have identified that such a new chemical structure can improve the shortcomings of the prior art in that the quantitative intensity is weakened and the quantitative accuracy is lowered, to strongly provide the quantitation signal in the tandem mass spectrometer, thereby completing the present invention. Further, the present inventors have studied the analytical method that can also be applied to the quadrupole ion trap mass spectrometer using the isobaric labeling agent provided by the present inventors, and then, identified the method wherein the analysis can be conducted via the quantitation signal ion having a high mass, and further identified a method that can quantify the relative quantity of peptides and proteins in all kinds of mass spectrometers including quadrupole ion trap mass spectrometer using this technology. Thereby, the present invention is finally completed.